The clinical characteristics of pediatric patients infected by SARS-CoV-2 Omicron variant and whole viral genome sequencing analysis

Pediatric population was generally less affected clinically by SARS-CoV-2 infection. Few pediatric cases of COVID-19 have been reported compared to those reported in infected adults. However, a rapid increase in the hospitalization rate of SARS-CoV-2 infected pediatric patients was observed during Omicron variant dominated COVID-19 outbreak. In this study, we analyzed the B.1.1.529 (Omicron) genome sequences collected from pediatric patients by whole viral genome amplicon sequencing using Illumina next generation sequencing platform, followed by phylogenetic analysis. The demographic, epidemiologic and clinical data of these pediatric patients are also reported in this study. Fever, cough, running nose, sore throat and vomiting were the more commonly reported symptoms in children infected by Omicron variant. A novel frameshift mutation was found in the ORF1b region (NSP12) of the genome of Omicron variant. Seven mutations were identified in the target regions of the WHO listed SARS-CoV-2 primers and probes. On protein level, eighty-three amino acid substitutions and fifteen amino acid deletions were identified. Our results indicate that asymptomatic infection and transmission among children infected by Omicron subvariants BA.2.2 and BA.2.10.1 are not common. Omicron may have different pathogenesis in pediatric population.

Illumina whole genome amplicon sequencing. Extracted RNA was first reverse transcribed to cDNA and subjected to whole-genome targeted amplification of SARS-CoV-2 sequences using COVIDSeq TM SARS-CoV-2 Primer Panel (Illumina, USA), which contain primer sequences defined by the ARTIC network (ARTIC v4.1). The resulting~400bp amplicons were fragmented, end repaired, and A-tailed using COVIDSeq TM Library Prep Kit (Illumina, USA) according to manufacturer's instruction. Unique dual indexes were added to the samples using the 10 base pair index i7 and i5 adapters of COVIDSeq TM (Illumina, USA) according to manufacturer's instruction. The size distributions of the sequencing libraries were checked using Tapestation 4150 (Agilent, USA). KAPA Pure Beads were used to perform double size selection of the libraries and equimolar pooling of libraries was performed by quantifying the samples using Qubit 4 fluorometer (Thermo Fisher, USA). Libraries were sequenced using NextSeq2000 P2 2 x 100bp kit (Illumina, USA) with at least 10M reads per sample.
Bioinformatic analysis. Sequencing data was analyzed using DRAGEN COVID Lineage (version 3.5.9; Illumina Inc., USA) and method as described previously [11,12]. In brief, sequencing reads of human origin were removed using NCBI Human Read Scrubber algorithm. ARTIC primer sequences were removed from the reads, followed by aligning to the reference genome Wuhan-Hu-1 (GenBank accession number MN908947.3) using DRAGEN (Illumina Inc., USA). Samples with less than 90 amplicons detected are filtered. Variant calling and consensus genome assembly with respect to the reference genome were performed using DRAGEN (Illumina Inc., USA) using default parameters. Nucleotide and amino acid positions were numbered according to the reference genome. All genome sequences analyzed in this study were submitted to GISAID under accession numbers EPI_ISI_13822514 to 13822565 and EPI_ISI_14336314 to 14336324. Pangolin lineages of the consensus genomes were assigned using Pangolin COVID-19 Lineage Assigner (version 4.0.2) [13]. Phylogenetic clades of the consensus genomes were mapped using Nextclade (version 1.11.0) [14].

Results
A total of sixty-eight laboratory-confirmed SARS-CoV-2 infected children aged between 0 and 17 years old were studied (mean Ct value: 18.5 ± 5.2 obtained by cobas 1 Liat 1 SARS-CoV-2 & Influenza A/B assay) (Fig 1). Among these cases, ten cases (14.7%) were in children aged below 1 year old, twenty-one cases (30.9%) were in children aged between 1 and 2 years old, fifteen cases (22.1%) were in children aged between 3 and 5 years old, ninteen cases (27.9%) were in children aged between 6 and 12 years old and three cases (4.4%) were in children aged between 13 and 17 years of age ( Table 1).

Clinical presentation
Results of this study suggest a milder course of disease in children infected by Omicron subvariants BA.2.2 and BA.2.10.1. However, no asymptomatic case was identified. Fifty-one children (75%) presented more than one sign or symptom while seventeen children (25%) presented one sign or symptom only. No case of co-infection was found. The most common symptoms reported were fever (94.1%), cough (33.8%), running nose (33.8%), sore throat (32.4%) and vomiting (23.5%). Two children (2.9%) had diarrhea and one child (1.5%) had shortness of breath (Table 1, Fig 2). Children in this cohort infected by Omicron (Fig 3). Among all the 68 pediatric cases of SARS-CoV-2 infection, none of them displayed any underlying conditions.

Transmission
In nine cases (13.2%), cohabiting family members were reported to have similar symptoms as the infected children. Two cases (2.9%) were reported to have a contact history with suspected or confirmed COVID-19 cases in the past 21 days. None of the cases had travel history outside Hong Kong in the past 21 days. This indicates that all the sixty-eight pediatric COVID-19 cases were local infections.

Phylogenetic analysis
Two distinctive groups were identified. The first group consisted of four genome sequences that belong to subvariant BA.2.10.1 whereas the second group consisted of sixty-one genome sequences that belong to subvariant BA.2.2 (Fig 4).

Viral genome mutations analysis
Compared to the reference genome of Wuhan-Hu-1 strain, a total of one nucleotide insertion, one frameshift mutation, one hundred and eighteen nucleotide substitutions, four nucleotide deletions and seven PCR primers and probes changes were identified. On protein level, eightythree amino acid substitutions and fifteen amino acid deletions were identified in the B.1.1.529 Omicron variant ( Table 2).

Travel History
With travel history outside Hong Kong in past 21 days

Contact history
Contacted with suspected / confirmed COVID-19 cases in past 21 days

PLOS ONE
Nucleotide insertion. One nucleotide insertion (14760T) was found in nine sequenced subjects at the non-structural protein 12 (NSP12) of the open reading frame 1b (ORF1b), leading to a novel frameshift mutation.
PCR primers and probes changes. A total of seven polymerase chain reaction (PCR) primers and probes changes in WHO listed SARS-CoV-2 primers and probes were found in Omicron subvariants BA.2.2 and BA.2.10.1 ( Table 4). Of the seven changes, five (71.4%) were located in N gene. C28770T was located within the N gene probe of Charité, G28881A,

PLOS ONE
Whole viral genome sequencing analysis of SARS-CoV-2 Omicron variant in pediatric patients G28882A and G28883C were located within the N gene forward primer of China CDC, whereas C28311T was located within the N1 gene probe of US CDC. One change (14.3%) was located in RdRp gene. T15521A was located within the RdRp gene reverse primer of Charité. One (14.3%) was located in E gene. On the other hand, C26270T was located within the E gene forward primer of Charité.

Discussion
To understand the epidemiology, infection risk, severity and viral genome of SARS-CoV-2 in children, a total of sixty-eight SARS-CoV-2 genome sequences collected from pediatric patients between January 2022 and May 2022 were determined by whole viral genome amplicon sequencing using Illumina next generation sequencing platform, followed by phylogenetic analysis. The more commonly presented symptoms in children infected by Omicron variant were fever, cough, running nose, sore throat and vomiting. Of the sixty-eight pediatric cases, fifty-one children (75%) presented more than one sign or symptom while seventeen children (25%) presented one sign or symptom only. No asymptomatic cases were identified. The cycle threshold (Ct) values in the sixty-eight pediatric cases were low (mean Ct value: 18.5 ± 5.2), showing that the viral load was high in these cases and these infected children were infectious to others. The data suggests that asymptomatic infection and transmission among children infected by Omicron    children aged above 3 were recommended to receive COVID-19 vaccination. In this cohort, thirty-one children (45.6%) were aged below 3. Of the other thirty-seven children (54.4%) aged above 3, six (16.2%) of them have received COVID-19 vaccination. The pediatric population was generally less affected clinically by SARS-CoV-2 infection in the past few waves of COVID-19 outbreaks caused by variants other than Omicron. This discrepancy in clinical responses between adults and pediatric population may have been due to the differences in various immune responses and physiological differences such as lower ACE2 expression in children relative to adults [4], elevated baseline IgM targeting coronavirus antigens [5] and stronger early innate antiviral immune responses in children [6]. However, during this Omicron variant (B.1.1.529) dominated fifth wave of COVID-19 outbreak in Hong Kong, there was a rapid increase in the hospitalization rate in SARS-CoV-2 infected pediatric patients. In some rare cases (0.002%) of the pediatric cases, some children developed multisystem inflammatory syndrome (MIS-C) after being infected by the SARS-CoV-2 virus [15,16].
In a recent study that compared the antibody specificity in COVID-19 pediatric population to that in adults, the antibodies response against the structural protein E and accessory protein ORF8 was found to be significantly elevated in SARS-CoV-2 infected pediatric population, compared to adults [16]. In contrast, antibodies against structural proteins S1 and M and accessory proteins ORF3a and ORF7b were found to be reduced compared to adults [16]. From our whole viral genome analysis, no amino acid mutations were found in the accessory proteins ORF8 and ORF7b of the SARS-CoV-2 Omicron subvariants BA.2.2 and BA.2.10.1 in the infected pediatric population. Pediatric population might be more susceptible to SARS-CoV-2 Omicron variant ORF8 wildtype. SARS-CoV-2 ORF8 is a 121-amino acid long accessory protein that mainly acts as immune-modulator to down-regulate MHC class I molecules in order to shield the infected cells from cytotoxic T cells [16][17][18]. On the other hand, ORF8 is a potent inhibitor that suppresses type I interferon responses [17][18][19]. ORF8 modulates both adaptive host immunity and innate immune responses and studies have demonstrated that ORF8 is associated with the severity of COVID-19 [17,20]. One amino acid substitution (T9I) was found at the transmembrane region of E proteins [21]. However, no association between this mutation and the transmission and severity of COVID-19 could be identified. Three amino acid substitutions (Q19E, A63T and K162Q) were found in M protein, twenty-five amino acid substitutions (T19I, A27S, G142D, N188I, V213G, G339D, S371F, S373P, S375F,  T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y,  Y505H, D614G, H655Y, N679K, P681H) and three amino acid deletions (L24-, P25-and P26-) were found in the S1 subunit of S protein and two amino acid substitutions (T223I and T269M) were found in ORF3a. ORF3a regulates the apoptosis and inflammatory responses in the infected cells. ORF3a can also activate the innate immune signaling receptor NLRP3 inflammasome, which results in tissue inflammation and cytokine production [22]. In addition to the above structural and accessory proteins, from our viral genome analysis, one amino acid substitution (P10S) and three amino acid deletions (E27-, N28-and A29-) were found in the ORF9b of Omicron variant. SARS-CoV-2 ORF9b can form a complex with a mitochondrial import receptor, Tom70. Recent studies have demonstrated that this complex can modulate the host immune response by compromising type I interferon synthesis [23,24]. However, whether these mutations in ORF9b can enhance the deleterious functions of ORF9b of Omicron variant to case infection in pediatric population, remains to be determined by further functional studies.
In this study, a novel frameshift mutation caused by insertion (14760T) was found at the NSP12 of the ORF1b region. NSP12 is a 932-amino acid long protein. It is an essential RNAdependent RNA polymerase (RdRp) of the replication transcription complex (RTC) responsible for making copies of genomic and subgenomic RNAs and polymerizing antisense RNA during viral replication of SARS-CoV-2 [25]. During the replication of SARS-CoV-2 genome, NSP12 binds to its cofactors NSP7 and NSP8 to activate its capability to replicate long RNA [26]. NSP8 can initiate the replication process and operate as a primase [27,28]. NSP7 participates in the viral replication process by binding to NSP12 as another primase [27]. Therefore, NSP12-NSP7-NSP8 complex is one of the potential targets for the development of antiviral agents and COVID-19 therapies. However, no association between this frameshift mutation identified in this study and the transmission and severity of COVID-19 can be identified so far. Other than that, no novel mutations were found in the genome of the SARS-CoV-2 Omicron variant isolated from infected pediatric population. There was also no significant difference in the genomes of the SARS-CoV-2 Omicron variant infecting the pediatric population, compared to those of the infecting adults. Without any specific mutations in the genome of SARS-CoV-2 infecting pediatric population, these findings and the difference in the distribution of antibodies to structural proteins and accessory proteins of SARS-CoV-2 detected by Hachim et al. [16] support the hypothesis that SARS-CoV-2 Omicron variant may have different pathogenesis in pediatric population, when compared to adults and other variants. Another possible reason for the increasing number of infected pediatric population during Omicron variant-dominated outbreak may be the altered TMPRSS2 usage by SARS-CoV-2 Omicron variants [29,30]. One of reasons that children were less affected clinically by SARS-CoV-2 was due to lower ACE2 and TMPRSS2 expression compared to adults. However, with over thirty mutations in the S protein, the S protein of Omicron variant was found to be less efficiently cleaved compared to other variants such as Alpha and Delta [29]. The less efficient cleavage of S protein and defect in the entry of Omicron variant promotes the change in the use of TMPRSS2 for viral entry and hence altering the entry pathway of Omicron variant to the cathepsin-dependent endosomal route, which is TMPRSS2 independent [29]. A high ratio of C-to-T nucleotide mutations were found (40.7%) in the genome of Omicron variant. It could be the results of host-driven antiviral editing [31]. We have also identified a total of seven PCR primers and probes changes in WHO listed SARS-CoV-2 primers and probes in Omicron subvariants BA.2.2 and BA.2.10.1 in this study. C28770T locates within the N gene probe of Charité, G28881A, G28882A and G28883C locate within the N gene forward primer of China CDC, and C28311T locates within the N1 gene probe of US CDC. T15521A locates within the RdRp gene reverse primer of Charité. C26270T locates within the E gene forward primer of Charité. These mutations in the target regions of PCR primers and probes could result in false negative results during PCR detection. Nucleic acid amplification tests (NAAT) such as real-time reverse transcription polymerase chain reaction (rRT-PCR) are still the preferred method for SARS-CoV-2 detection worldwide. Therefore, it is suggested that dual targets are crucially important to minimize the risk of false negative results of detecting Omicron variant [32,33]. Considering the primer and probe binding site changes as the virus evolve, RT-PCR with dual target design is an important strategy to improve assay robustness to mutations in primer and probe binding sites. Some of the mutations in primer and probe sites may cause binding efficiency to drop hence could yield false negative results [34]. Examples of dual target like assays developed by Tombuloglu et al. targeting viral RdRP gene, viral E gene [32] or N gene [33] and human RNase P gene as internal control. NAAT such rRT-PCR assays are the standard method for SARS-CoV-2 detection worldwide, for their high sensitivity and specificity. Novel molecular assays were developed to detect SARS-CoV-2 viruses in the attempt to produce assays that do not rely on specialized molecular laboratory environment to perform. These assays improve accessibility of molecular tests in aid of better contact tracing efforts. Examples of such assays are CRISPR-Cas protein based, and reverse transcription loop-mediated isothermal amplification (RT-LAMP) methods. One-pot formats of these assays could be performed on a heat-block or water bath and be used in resource-poor situations.
CRISPR-Cas9 methods first amplify SARS-CoV-2 RNA in samples with isothermal amplification, then detect by gRNA binding to SARS-CoV-2 genome and the Cas protein cleave reporter RNA strand to give a signal. CRISPR-Cas related methods could detect SARS-CoV-2 at 10-100 sequences per microliter of sample [35] and lateral flow strips to detect end point. RT-LAMP assays targeting N gene, RdRp gene, S and E genes were made with the possibility to use colorimetric detection with pH indicators [36], which could remove the need of fluorometric instruments. These novel methods could be further implemented in areas with less access to complex instruments.
From this study, we also found that fever (94.1%), cough (33.8%), running nose (33.8%), sore throat (32.4%) and vomiting (23.5%) were the more commonly reported symptoms in children infected by Omicron variant. For adults who were infected by Omicron variant, headache (76.5%), running nose (74.9%), sneezing (69.3%) and sore throat (68.4%) were the more commonly reported symptoms. Only 39.2% and 18.7% of the infected adults presented fever and diarrhoea respectively [37]. There is no evidence to show that children infected by Omicron subvariant BA.2.10.1 would result in worse and more severe clinical outcomes than children infected by BA.2.2 and no sexual dimorphism was observed. Our SARS-CoV-2 Omicron subvariants BA.2.2 and BA.2.10.1 genome sequences enriched the understanding of SARS-CoV-2 mutational landscape and improved the understanding of SARS-CoV-2 infection in pediatric population. The strengths of this study include a high average sequencing depth and a high average genomic coverage of the whole viral genome sequencing. Host-derived DNA sequences were removed to allow greater sequencing depth of the viral genome. The limitations of this study are that Sanger sequencing was not performed to confirm the mutations identified. Also, our cohort did not include any cases of the children with MIS-C.

Conclusion
In conclusion, asymptomatic infection and transmission among children infected by Omicron subvariants BA.2.2 and BA.2.10.1 are not common. A novel frameshift mutation was found in the ORF1b region (NSP12) of the genome of Omicron variant in this study. There was also no significant difference between the genomes of the SARS-CoV-2 Omicron variant infecting the pediatric population, and those infecting the adults. However, Omicron variant may have different pathogenesis in pediatric population, when compared to adults.